PROJECT SUMMARY To maintain ongoing homeostasis, hematopoietic stem cells (HSCs) possess elaborate mechanisms to balance self-renewal, proliferation and differentiation. During development and after infection HSCs adjust self-renewal versus differentiation to expand to meet but not overshoot the needs of the organism. Understanding how HSCs modulate this balance is critical for manipulating HSCs ex vivo for improved use in transplantation. To improve our understanding balancing HSC self-renewal with other needs, we have studied molecular pathways connected to the proto-oncogene, MLL1, due to its unique ability to regulate an HSC-enriched transcriptional program and potential to become leukemogenic upon chromosomal translocation in acute leukemia. We have shown using mouse loss- of-function models that Mll1 is essential for the development of HSCs during embryogenesis and for their maintenance in adult bone marrow. This role is carried out by maintaining expression of a network of transcriptional regulators including Hoxa9, Mecom/Evi1, Prdm16 and Meis1. Therefore we hypothesize that increasing MLL1 protein levels or activity could coordinately enhance this transcriptional network thereby enhancing HSC self-renewal and preventing differentiation. To test this hypothesis, we have created a new animal model that for the first time overcomes the resistance of many cell types to overexpress wild-type MLL1. Using single-copy integration into a doxycycline- inducible locus, we show increased hematopoietic output from embryoid bodies and embryo progenitors upon hMLL1 induction. In this Single Aim Shine II proposal, we seek to evaluate this animal model in detail to determine a) the developmental and differentiation stages that are permissive for hMLL1-induced hematopoietic expansion and b) identify regulatory networks that are altered by increased levels of MLL1 in relevant hematopoietic populations. It is our expectation that generating this proof-of-principle data linking increased MLL1 to enhanced hematopoiesis and determining the underlying mechanisms will guide future efforts to experimentally manipulate MLL1 in human stem and progenitor cells to expand their numbers and improve function in transplantation settings.